Multiplexing methods for indentifying nucleic acids using denaturing liquid chromatography

ABSTRACT

A method is provided for the separation of nucleic acid samples. The method includes: providing a test mixture of a plurality of nucleic acid samples, wherein each sample is labeled with a spectrally detectable tag; applying the test mixture to a reversed phase solid support; eluting the mixture under partially denaturing conditions; and detecting spectrally resolved signals produced by the nucleic acid samples labeled with the detectable tags.

FIELD OF THE INVENTION

[0001] The present invention relates to multiplexed chromatographicmethods. Such methods can be used for detecting mutations within apopulation of nucleic acid samples. They can also be used for genotypingand haplotyping.

BACKGROUND OF THE INVENTION

[0002] Deciphering the genetic code and the establishment of thestructure of deoxyribonucleic acid (DNA) in the early 1960s initiated arevolution in modern biology. Since that time, numerous methods havebeen developed for the isolation, analysis, and manipulation of nucleicacid samples.

[0003] One such method developed for the analysis of nucleic acidsamples is polymerase chain reaction amplification (referred to hereinas “PCR”). PCR is an in vitro method for replicating a defined (ortarget) DNA molecule to increase the amount of total DNA for subsequentanalysis, such as sequencing, Northern and Southern hybridizations, andthe like. Typically, the amount of total DNA increases exponentially,i.e., it is amplified. Thus, PCR can be utilized in connection with avariety of techniques when it is desirable to manipulate and analyzegenetic information of a DNA molecule that may be in low copy numbers.For example, PCR may be used in connection with cloning genes,sequencing, genome mapping, site directed mutagenesis, diagnosticassays, environmental monitoring, to name a few.

[0004] Due to the vast amount of genetic information that is capable ofbeing generated and gathered, intense efforts are underway to developnew and faster methods of DNA detection, sizing, quantification,sequencing, and gene identification including the mapping of humandisease genes. Although the efficiency of these processes has beenimproved by automation, more efficient and less expensive methods muststill be developed to efficiently carry out genomic-scale DNA analyses.

[0005] The detection of polymorphisms is becoming increasinglyimportant, particularly in gene mapping. Although the majority of DNA inhigher organisms is identical in sequence among the chromosomes ofdifferent individuals, a small fraction of DNA is variable orpolymorphic in sequence. It is this variation which is the essence ofgenetic science and human diversity.

[0006] Mutations arise either due to environmental effects or randomlyduring replication as a change in the sequence of a gene, with differentmutations having differing consequences. In fact, single base pairchanges, called single nucleotide polymorphisms (SNPs) are frequent inthe human genome. The level of genetic variation between two individualsequences is estimated to be on average one difference per 1,000 basepairs. Based on this estimate, the average amount of genomic variationbetween individuals is about 3 million base pairs. It is this normalpolymorphism, which provides the basis for some of the emerging genelocalization strategies.

[0007] As the sequences of greater numbers of genes are identified, thedetection of specific polymorphisms in such genes and the correlation tospecific diseases can provide an invaluable tool in the screening anddetection of diseases. Diagnostic screening methods for polymorphismsare also useful in the detection of inherited diseases in which either asingle point mutation or a few known mutations account for all cases(e.g., sickle cell disease). Presently, over 200 genetic disorders canbe diagnosed using recombinant DNA techniques.

[0008] Such techniques have also been used for other purposes, such asfor forensic screening.

[0009] Presently used methods for screening for polymorphic sites withina gene include single-stranded conformation polymorphism (SSCP),denaturing gradient gel electrophoresis (DGGE), RNase A cleavage,chemical cleavage, allele specific oligonucleotides (ASOs), ligasemediated detection of mutations, and denaturing high performance liquidchromatography.

[0010] Briefly, in single-stranded conformation polymorphism (SSCP), DNAis denatured and then immediately run on a non-denaturing gel. Thesecondary structures of wild-type strands or mutant single strandsdiffering by a single base are usually sufficiently different to resultin different migration rates on polyacrylamide gels.

[0011] In denaturing gradient gel electrophoresis (DGGE), eitherhomoduplex or heteroduplex double stranded DNA is electrophoresed underdenaturing conditions of increasing concentration until the last domainis denatured, and migration of the DNA through the gel is retarded. DNAsequences differing by a single base pair migrate at different ratesalong the gel, thereby allowing detection of a polymorphic site, ifpresent.

[0012] RNase A cleavage utilizes the enzyme ribonuclease A to cutRNA-DNA hybrids wherever there is a mismatch between a nucleotide in theRNA strand and the corresponding nucleotide in the DNA strand. Thechemical cleavage method is based upon a similar principle but useshydroxylarnine and osmium tetroxide to distinguish between mismatched Cor T nucleotides, respectively. The position of the mismatch (e.g., themutation) is defined by sizing on gel electrophoresis after cleavage atthe reactive position by piperidine.

[0013] Allele-specific oligonucleotide probes (ASOs) are probes that aredesigned to hybridize selectively to either a normal or a mutant allele,where the probes are developed to distinguish between the normal andmutant sequence. This is done by altering the stringency ofhybridization to a level at which each of the oligonucleotides willanneal stably only to the sequence to which it is perfectlycomplementary and not to the sequence with which it has the singlemismatch.

[0014] The ligase-mediated method for detecting mutations makes use ofthe fact that the ends of two single strands of DNA must be exactlyaligned for DNA ligase to join them. In utilizing this technique,oligonucleotides complementary to the target sequence, 5′ to andincluding the mutation site, are synthesized and labeled. A thirdoligonucleotide complementary to the common sequence 3′ to the mutationsite is synthesized and also labeled. The oligonucleotides are thenhybridized to strands of the target. In cases in which the 5′ and 3′oligonucleotides form a flush junction that can be joined by DNA ligase,ligation occurs. However, a single base pair mismatch occurring betweenthe normal 5′oligonucleotide and the mutation site is sufficient toprevent the ligase from acting and can readily be detected.

[0015] A common approach to analysis of DNA polymorphisms relies onvariations in the lengths of DNA fragments produced by restrictionenzyme digestion. The polymorphisms identified using this approach aretypically referred to as restriction fragment length polymorphisms orRFLPs. Polymorphisms involving variable numbers of tandemly repeated DNAsequences between restriction enzyme sites, typically referred to asmicrosatellites or variable numbers of tandem repeats (VNTRs), have alsobeen identified.

[0016] While existing methods may locate polymorphic sites, pointmutations, insertions and deletions on a gene, many of these methods aregenerally time consuming, necessitate multiple handling steps, requireproduct purification, are not readily adaptable to automation, havelimitations in sensitivity and accuracy, and are typically limited todetection in small-sized nucleic acid fragments.

[0017] Furthermore, existing methods typically do not yield haplotypeinformation (i.e., linked polymorphism) without the use of multiple, andoften complicated, steps that may incorporate toxic chemicals. See, forexample, Verpy et al., Proc. Natl. Acad. Sci., USA, 95, 1873-1877(1994).

[0018] Denaturing high performance liquid chromatography for separatingheteroduplex (double-stranded nucleic acid molecules having less than100% sequence complementarity) and homoduplex (double-stranded nucleicacid molecules having 100% sequence complementarity) nucleic acidsamples (e.g., DNA or RNA) in a mixture is described in U.S. Pat. No.5,795,976 (Oefner et al.). In the separation method, a mixturecontaining both heteroduplex and homoduplex nucleic acid samples isapplied to a stationary reversed phase support. The sample mixture isthen eluted with a mobile phase containing an ion-pairing reagent and anorganic solvent. Sample elution is carried out under conditionseffective to at least partially denature the duplexes and results in theseparation of the heteroduplex and homoduplex molecules. Also disclosedis the amplification of homoduplex and heteroduplex molecules using thepolymerase chain reaction. The amplified DNA molecules are denatured andrenatured to form a mixture of heteroduplex and homoduplex moleculesprior to separating the molecules. This method can be used to runmultiple samples at once as long as the different samples do notco-elute in time, which is referred to as multiplexing in time. Thedisadvantage of this is that each analytical run takes longer than asingle non-multiplexed run.

SUMMARY OF THE INVENTION

[0019] What is yet needed is a relatively rapid method for identifyingnucleic acids, specifically for distinguishing individual polymorphicnucleic acid molecules. What is also needed is a relatively rapid methodfor genotyping and haplotyping that involves relatively fewer steps, iscapable of automation, and generates information relatively quickly. Thepresent invention provides such methods. In a preferred embodiment, amethod of the present invention can be used to distinguish individualPCR amplicons (also referred to as PCR products herein) from a PCRreaction mixture. Significantly and advantageously, the presentinvention involves the use of multiplexed denaturing liquidchromatography, particularly multiplexed denaturing high performanceliquid chromatography.

[0020] As used herein, “multiplexing” or “multiplexed” refers to theability to run multiple (different) samples substantially simultaneouslyunder similar conditions and be able to reconstruct the dataindividually for each sample. It involves using a detectable label ortag that can be monitored spectrally. In essence, the method of thepresent invention involves spectral multiplexing. This is distinct frommultiplexing in time because in the present invention all samples can berun in the same time period in which one sample could be run (i.e.,substantially simultaneously). In a preferred embodiment of theinvention, using fluorescence multiplexing, the samples are exposed toradiation having a wide range of wavelengths, individual wavelengths aremonitored, and then the mixed signals, which are spectrally resolved,are reconstructed according to their spectral properties.

[0021] In one embodiment of the present invention, a method is providedfor separating nucleic acid (e.g., DNA and RNA) samples in a testmixture. The method includes: providing a test mixture of a plurality ofnucleic acid samples, wherein each sample is labeled with a spectrallydetectable tag; applying the test mixture to a reversed phase support;eluting the mixture under partially denaturing conditions to separate atleast one nucleic acid sample from the test mixture (preferably, all ofthe nucleic acid samples are separated from each other during theelution); and detecting spectrally resolved signals produced by thenucleic acid samples labeled with the detectable tags. Preferably, thenucleic acid samples include PCR products (i.e., PCR amplicons),particularly heteroduplexes and homoduplexes. Preferably, the reversedphase solid support is in a high performance liquid chromatography(HPLC) column.

[0022] Preferably, the tag is selected from the group of electromagneticand electrochemical tags, and more preferably, the tag is selected fromthe group of spectrophotometric and spectrofluorometric tags, and mostpreferably, the tag is a spectrofluorometric tag. In a particularlypreferred embodiment, the detectable label or tag is a fluorescent dye.Preferably, the test mixture is formed by combining a differentfluorescent dye with each nucleic acid sample to form a labeled nucleicacid sample; and combining the labeled nucleic acid samples to form thetest mixture. Preferably, a different fluorescent dye is added to eachnucleic acid sample separately during polymerase chain reactionamplification of the nucleic acid sample prior to combining the nucleicacid samples to form the mixture.

[0023] In the present method, the eluting step includes the use of amobile phase containing an ion-pairing agent and optionally an organicsolvent. Examples of ion-pairing agents include amines such as loweralkyl primary, secondary, and tertiary amines, ammonium salts such aslower trialkylammonium salts (e.g., triethylammonium acetate) and loweralkyl quaternary ammonium salts.

[0024] A variety of methods can be used for partial denaturation of themixture of nucleic acid samples (e.g., PCR amplicons). For example,temperatures of about 50° C. to about 80° C. can be used. Alternatively,a chemical reagent for denaturation can be used in the mobile phase.

[0025] Preferably, detecting spectrally resolved signals involves theuse of spectrophotofluorometric methods of detection in which excitationand emission wavelengths can be independently chosen. In such methods,emission wavelengths can be detected at very low concentrations, oftenat less than about 1 nanomolar concentrations. Thus, in preferredembodiments, the methods of the present invention include detectingspectrally resolved signals using a spectrophotofluorometric HPLCdetector.

[0026] Typically, detecting spectrally resolved signals produced by thenucleic acid samples labeled with the detectable tags includes passingthe separately labeled nucleic acid samples through a detection zone ofa detector substantially simultaneously, each sample generating aspecific signal which is spectrally resolved from the specific signalsof the other nucleic acid samples. Preferably, the detector excites thedetectable tags at one wavelength and detects emissions at multiplewavelengths. Alternatively, the detector can excite the detectable tagsusing zero-order excitation.

[0027] In one preferred embodiment, the present invention provides amethod for detecting genotypic variations. The method includes:providing a pre-mixture that includes one unlabeled nucleic acid sampleand two or more reference genotypes of labeled nucleic acid, whereineach labeled nucleic acid is labeled with a different spectrallydetectable tag and the unlabeled nucleic acid is present in an excessamount relative to the total amount of labeled nucleic acid; denaturingand reannealing the pre-mixture to form a test mixture comprisinglabeled/unlabeled nucleic acid duplexes; applying the test mixture to areversed phase support; eluting the test mixture under partiallydenaturing conditions to separate at least one of the labeled/unlabelednucleic acid duplexes from the test mixture; detecting spectrallyresolved signals produced by the labeled nucleic acid to define anelution profile for each tag; and deducing from the elution profiles thecomposition of the unlabeled nucleic acid sample. Preferably, the tag isa spectrally detectable tag selected from the group of anelectromagnetic tag and an electrochemical tag.

[0028] In another preferred embodiment, the present invention provides amethod for determining haplotypes. The method includes: providing apre-mixture that includes one unlabeled nucleic acid sample and four ormore reference haplotypes of labeled nucleic acid, wherein each labelednucleic acid sample includes two or more alleles, each referencehaplotype is labeled with a different detectable tag, and the unlabelednucleic acid is present in an excess amount relative to the total amountof labeled nucleic acid; denaturing and reannealing the pre-mixture toform a test mixture comprising labeled/unlabeled nucleic acid duplexes;applying the test mixture to a reversed phase support; eluting the testmixture under partially denaturing conditions to separate at least oneof the labeled/unlabeled nucleic acid duplexes from the test mixture;detecting spectrally resolved signals produced by the labeled nucleicacid to define an elution profile for each tag; and deducing from theelution profiles the composition of the unlabeled nucleic acid sample.Preferably, the tag is a spectrally detectable tag selected from thegroup of an electromagnetic tag and an electrochemical tag.

DEFINITIONS

[0029] The following terms, as used herein, have the meanings asindicated:

[0030] “Reversed phase support” refers to a stationary support(including the base material and any chemically bonded phase) for use inliquid chromatography, particularly high performance liquidchromatography (HPLC), which is less polar (e.g., more hydrophobic) thanthe starting mobile phase.

[0031] “Ion-pair (IP) chromatography” refers to a chromatographic methodfor separating samples in which some or all of the sample componentscontain functional groups which are ionized or are ionizable. Ion-pairchromatography is typically carried out with a reversed phase column inthe presence of an ion-pairing reagent.

[0032] “Ion-pairing reagent” is a reagent which interacts with ionizedor ionizable groups in a sample to improve resolution in achromatographic separation. An “ion-pairing agent” refers to both thereagent and aqueous solutions thereof. An ion-pairing agent is typicallyadded to the mobile phase in reversed phase liquid chromatography foroptimal separation. The concentration and hydrophobicity of anion-pairing agent of choice will depend upon the number and types (e.g.,cationic or anionic) of charged sites in the sample to be separated.

[0033] “Homoduplex molecules” are typically composed of twocomplementary nucleic acid strands.

[0034] “Heteroduplex molecules” are typically composed of twocomplementary nucleic acid strands (e.g., DNA or RNA), where the strandshave less than 100% sequence complementarity. Functionally, in a mixedpopulation of homoduplex and heteroduplex molecules, shorter strands(e.g., typically about less than 50 base pairs in size) of heteroduplexmolecules elute as peaks corresponding to their respective denaturedsingle strands under select denaturing conditions using reversed phaseion-pairing chromatography, separable from those of homoduplexmolecules. In a mixed population of homoduplex and heteroduplexmolecules larger than about 50 base pairs in length, heteroduplexmolecules typically elute with shorter retention times than those ofhomoduplexes of essentially the same size under select denaturingconditions using reversed phase ion-pairing chromatography.

[0035] A heteroduplex molecule that is “partially denatured” under agiven set of chromatographic conditions refers to a molecule in whichseveral complementary base pairs of the duplex are not hydrogen-bondpaired, such denaturing typically extending beyond the site of thebase-pair mismatch contained in the heteroduplex, thereby enabling theheteroduplex to be distinguishable from a homoduplex molecule ofessentially the same size. In accordance with the present invention,such denaturing conditions may be either chemically (e.g., resultingfrom pH conditions) or temperature-induced, or may be the result of bothchemical and temperature factors.

[0036] “Genotype” refers to the genetic constitution of a cell or anorganism such that expression of the genetic constitution gives rise toan organism's physical appearance. In general, the genetic constitutionmeans a gene, wherein alternative forms of the gene are called“alleles.” Typically, an allele is carried at a genetic locus, orposition, on an organism's chromosome. “Genotyping” refers to detectingwhich alleles are present in a given individual.

[0037] “Haplotype” refers to a set of closely linked alleles on aspecific chromosome carried by an individual and inherited as a unit,such as the alleles of the major histocompatibility complex on humanchromosome number 6. “Haplotyping” refers to detecting a change ormutation that may be within one or all of the linked alleles (i.e., thehaplotype).

[0038] “Primer” refers to an oligonuleotide, whether occurring naturallyas in a purified restriction digest or produced synthetically, which iscapable of acting as a point of initiation of synthesis when placedunder conditions in which synthesis of a primer extension product thatis complementary to a target nucleic acid strand is induced, i.e., inthe presence of nucleotides and an agent for polymerization (such as aDNA polymerase) and at a suitable temperature and pH. The primer ispreferably single stranded for maximum efficiency in amplification.Preferably, the primer is an oligodeoxyribonucleotide. The primer mustbe sufficiently long to prime the synthesis of extension products(referred to herein as “PCR products” and “PCR amplicons”) in thepresence of the polymerization agent. Primers are preferably selected tobe “substantially” complementary to a portion of the target nucleic acidsequence to be amplified. This typically means that the primer must besufficiently complementary to hybridize with its respective portion ofthe target sequence. For example, a primer may include anon-complementary nucleotide portion at the 5′ end of the primer, withthe remainder of the primer being complementary to a portion of thetarget sequence. Alternatively, non-complementary bases or longersequences can be interspersed into the primer, provided that the primersequence has sufficient complementarity with a portion of the targetsequence to hybridize therewith, and thereby form a template forsynthesis of the extension product.

BRIEF DESCRIPTION OF THE FIGURES

[0039]FIG. 1 shows the spectra of the fluorescence multiplexeddenaturing high performance liquid chromatography (DHPLC) runs of twoheteroduplexes.

DETAILED DESCRIPTION OF THE INVENTION

[0040] The present invention provides a method for separating nucleicacid samples (DNA and RNA), preferably generated from PCR amplification,using multiplexed denaturing liquid chromatography and moreparticularly, denaturing high performance liquid chromatography. Themethod can be utilized for detecting nucleic acid PCR products frommultiple amplification reactions, so long as each amplification reactionincorporates an agent having a unique spectral signature, wherein eachreaction does not contain the same agent.

[0041] The present invention also provides a method for genotyping andhaplotyping nucleic acid samples, preferably generated from PCRamplification, using multiplexed denaturing liquid chromatography andmore particularly, denaturing high performance liquid chromatography.

[0042] Multiplexing refers to a method of analyzing samplessubstantially simultaneously using some means by which the signals ofthe individual samples can be reconstructed separately and independentlyof the others. The advantage of multiplexing is that it allows moresamples to be analyzed in the same amount of time, increasing effectivesample throughput. For example, if multiple samples, labeled withspecific tags are separated simultaneously by chromatographic methods,the elution profile passing through the detector could be separated intothe individual original samples by using the detection characteristicsof the specific tags to generate specific detection channels.

[0043] Multiplexing is significant and advantageous because it addressesthe problem of the low throughput of liquid chromatography, particularlyDHPLC, by multiplexing the analysis using a detectable label or tag,preferably, a fluorescent dye. Furthermore, in preferred embodiments,use of PCR primers with different tags, such as fluorescent dyes, toamplify nucleic acid from different individuals allows the co-injectionduring a single run of multiple samples, thereby increasing thethroughput of the analysis.

[0044] Prior to the method of the present invention, the only methodavailable for multiplexing DHPLC was to co-inject PCR amplicons whichare sufficiently different in length to ensure that the peak patterns ofthe individual samples do not overlap in the chromatogram. This has verylimited utility since it only increases the throughput by a factor oftwo. Furthermore, it is not always possible to predict exactly wheredifferent PCR products will elute, especially the split peaks ofheteroduplexes.

[0045] PCR Amplification and Labeled Primers

[0046] PCR amplification involves separating two strands of nucleic acidand annealing oligonucleotide primers to a target nucleic acid moleculeand extending the nucleic acid molecule by nucleotide addition from theprimers by the action of a polymerase enzyme. The target molecule isdefined by 3′- and 5′-flanking nucleic acid portions to whicholigonulceotide primers are annealed. The primers are then extendedacross the target DNA molecule by using a heat-stable polymerase (suchas Taq DNA polymerase) in the presence of free deoxynucleosidetriphosphates (dNTPs), resulting in a double replication of the startingtarget nucleic acid molecule.

[0047] Preferably, PCR amplification generates PCR amplificationproducts (also referred to as PCR amplicons) incorporating a detectablelabel or tag. Thus, PCR amplification of target nucleic acid ispreferably accomplished by utilizing at least one primer containing adetectable tag. Preferably, each primer utilized in the amplificationreaction is labeled with a different spectrally detectable tag.Detectable tags are chosen such that they preferably behave similarly inliquid chromatography. That is, they are chosen such that they areretained on the reversed phase support for substantially the same amountof time. However, such tags also should produce spectrally resolvablesignals. This can be accomplished, for example, by selected fluorescentdyes in the same family such as Cy3 and Cy5, which are cyanine dyes,obtained from Amersham-Pharmacia Biotech.

[0048] In general, a spectral detection method (i.e., one in which thesignal generated can be restricted to a defined subset of a large numberof different physical possibilities), preferably an electromagnetic orelectrochemical technique, more preferably a spectrophotometric andspectrofluorometric technique, and most preferably, aspectrofluorometric technique, can be used, providing that the followingconditions are met. The signals produced by the labeled nucleic acidsamples can be resolved with little or no spectral overlap such that thepresence of a labeled sample doesn't produce a signal under thedetection conditions for other labeled samples. Also, the introductionof different detectable tags does not substantially perturb thedenaturation conditions of the nucleic acid samples. That is,introduction of a tag does not cause any significant reduction in theamount of nucleic acid denatured when compared to the amount denaturedunder the same conditions without the detectable tag. Further, thespectral detection system is amenable to on-line chromatographicdetection, and the tags do not substantially perturb the chromatographicretention characteristics of the nucleic acid samples. That is, evenwith the introduction of a tag the nucleic acid samples show at leastpartial separation between homoduplexes and heteroduplexes.

[0049] For example, ultraviolet, visible, or infrared absorbing tagscould be used that would produce specific resolvable ultraviolet,visible, or infrared signals. Alternatively, they may be labeled withtags that are detectable using electrochemical and nuclear magneticresonance. Nuclear Magnetic Resonance tags could be used that introduceresolvable chemical shifts. Electroactive groups could be used thatgenerate specific resolvable redox signals in amperometic detectors.Mass spectral tags can also be used for certain embodiments, but theyare less desired.

[0050] Other suitable tags include energy transfer coupled dyes in whichprimers are labeled with a donor and an acceptor dye. Also,chemiluminescent systems can be utilized which are typically defined asthe emission of absorbed energy (typically as light) due to a chemicalreaction of the components of the system, including oxyluminescence inwhich light is produced by chemical reactions involving oxygen.

[0051] Examples of a wide variety of tags (a chemical moiety that isused to uniquely identify a nucleic acid of interest) are disclosed inInternational Publication No. WO 97/27325. These may be covalently boundto the nucleic acid or otherwise associated with the nucleic acid suchthat they elute simultaneously. If covalently bound tags are usedaccording to the present invention, they are not cleaved prior to thenucleic acid entering the detector.

[0052] Preferably, the detectable tag has an excitation and/or emissionwavelength. Particularly preferred such tags are fluorescent agents.These are typically dye compounds that emit visible radiation in passingfrom a higher to a lower electronic state, typically in which the timeinterval between adsorption and emission of energy is relatively short,generally on the order of about 10-8 to about 10-3 second. Suitablefluorescent compounds can include fluorescein, rhodamine, luciferin, aswell as a wide variety of others known to one of skill in the art (see,for example, the list of dyes available on the world wide web at“www.apbiotech.com/product/product_index.htrnl” or “www.pebio.com” or“www.probes.com”). The use of fluorescent dyes in the HPLC separation ofnucleic acids is disclosed by Oefner et al., Anal. Biochem., 223, 39-46(1994).

[0053] Denaturing Liquid Chromatography

[0054] Liquid chromatography, preferably high performance liquidchromatography (HPLC), generally refers to a technique for partitioninga sample, or more specifically the components of a sample, between amobile phase (typically containing an ion-pairing reagent) and astationary phase. In the present invention, a chromatographic methodutilizes conditions effective to partially denature duplexes duringsample elution to thereby enable the separation and identification ofdifferent nucleic acid molecules in a mixture.

[0055] Typically, denaturing high performance liquid chromatography isused to identify mutations based on the separation of heteroduplexmolecules containing a single base mismatch from homoduplex molecules.As an example, a PCR amplicon from a target sample is mixed with thesame amplicon generated from a wild-type reference sample and heated tomelt the existing homoduplexes and then allowed to slowly cool resultingin both homo- and heteroduplexes. The HPLC running temperature is chosensuch that partial denaturation begins to occur in the area around thepolymorphic mismatch. In this partially denatured region, theion-pairing reagent of the mobile phase, is not able to interact asefficiently between the phosphate backbone and the stationary phase.Thus, heteroduplexes and homoduplexes have different retention times andare separated upon elution.

[0056] A variety of methods can be used for partial denaturation ofnucleic acid molecules. Elevated temperatures can be used for carryingout the separation method of the invention as long as they are not sohigh that complete denaturation occurs. Such temperatures are preferablyabout 50° C. to about 80° C., and more preferably about 55° C. to about65° C. depending on the specific sequence of the nucleic acid.Alternatively, a chemical reagent for denaturation can be used in themobile phase. Examples of such chemical reagents includedimethylsulfoxide, urea, formamide, glycerol, and betaine.

[0057] Stationary Phase

[0058] In the method of the present invention, a test mixture containinga mixture of nucleic acid samples (preferably, resulting from PCRamplification, more preferably, a mixture of heteroduplex molecules andhomoduplex molecules) is applied to a stationary phase. Generally, thestationary phase is a reversed phase material (which can include a basematerial and a chemically bonded phase), which is hydrophobic and lesspolar than the starting mobile phase (i.e., the starting gradient in agradient elution mode). A variety of commercially available reversedphase solid supports may be utilized in the present nucleic acidseparation method as long as they are able to separate unlabeled nucleicacid molecules.

[0059] Reversed phase columns or column packing materials for use in theinvention are typically composed of inorganic or organic materials,which may or may not be functionalized, such as silica, cellulose andcellulose derivatives such as carboxymethylcellulose, alumina, zirconia,polystyrene, polyacrylamide, polymethylmethacrylate, and styrenecopolymers (e.g., a styrene-divinyl copolymer formed from (i) a styrenemonomer such as styrene, lower alkyl substituted styrene (in which thebenzene ring contains one or more lower alkyl substituents),alpha-methylstyrene and lower alkyl alpha-methylstyrene and (ii) adivinyl monomer such as C₄-C₂₀ alkyl and aryl divinyl monomers includingdivinylbenzene and divinylbutadiene).

[0060] A preferred stationary support is a wide pore silica-basedalkylated support. The base material composing the solid support istypically alkylated. “Alkylated” as used in reference to the solidsupport refers to attachment of hydrocarbon chains to the surface of thebase material of the solid support. The hydrocarbon chains may besaturated or unsaturated and may optionally contain additionalfunctional groups attached thereto. The hydrocarbon chains may bebranched or straight chain and may contain cyclic groups such ascyclopropyl, cyclopropyl-methyl, cyclobutyl, cyclopentyl,cyclopentylethyl, and cyclohexyl.

[0061] Alkylation of the base material prevents secondary interactionsand can improve the loading of the stationary phase with the ion-pairingreagent to promote conversion of the solid support into a dynamicanion-exchanger. Typically, the base material is alkylated to possessalkyl groups containing at least 3 carbon atoms, generally about 3 toabout 22 carbon atoms, and preferably contains about 4 to about 18carbon atoms. The alkylated solid support phase may optionally containfunctional groups for surface modification. The presence or absence ofsuch functional groups will be dictated by the nature of the sample tobe separated and other relevant operational parameters.

[0062] The stationary phase may also include beads having a particlesize of about 1 micron to about 100 microns. As used herein, theparticle size is determined by measuring the largest dimension of theparticle (typically, the diameter for a spherical particle).

[0063] A stationary phase for use in the present method typically haspores with sizes ranging from less than about 30 Angstroms in diameter(e.g., nonporous materials) up to about 1000 Angstroms in size.“Nonporous stationary support” refers to a solid support composed of apacking material having surface pores of a diameter that excludespermeation of sample compounds into the pore structure, typically ofless than about 30 Angstroms in diameter. In using nonporous polymericsupport materials, the relatively small pore size excludes many samplecompounds from permeating the pore structure and may promote increasedinteraction with the active surface. The stationary phase may alsocontain more than one type of pore or pore system, e.g., containing bothmicropores (less than about 50 Angstroms) and macropores (greater thanabout 1000 Angstroms). For achieving separations of samples containingheteroduplexes and homoduplexes of up to about 1000 base pairs in size,the stationary phase will preferably have a surface area of about 2 m²/gto about 400 m²/g, and more preferably about 8 m²/g to about 20 m²/g, asdetermined by nitrogen adsorption.

[0064] Commercially available stationary phases include a wide poresilica-based C18 material commercially available under the tradedesignation “ECLIPSE ds DNA” from Hewlett Packard Newport, Newport, DE,and an alkylated polystyrene-divinylbenzene nonporous materialcommercially available under the trade designation “DNASep” fromTransgenomic, San Jose, Calif.

[0065] Mobile Phase

[0066] The separation method of the present invention utilizesdenaturing liquid chromatography, preferably denaturing high performanceliquid chromatography (DHPLC), and more specifically, ion-pairingreversed phase HPLC (IP-RP-HPLC). In carrying out the separationaccording to the present method, the aqueous mobile phase contains anion-pairing agent and a solvent, preferably an organic solvent.

[0067] The selection of aqueous mobile phase components will varydepending upon the nature of the sample and the degree of separationdesired. Any of a number of mobile phase components typically utilizedin ion-pairing reversed phase HPLC are suitable for use in the presentinvention. Several mobile phase parameters (e.g., pH, organic solvent,ion-pairing reagent and counterion, and elution gradient) may be variedto achieve optimal separation, such as the percent organic solvent,temperature, and concentration of the components.

[0068] Ion-pairing reagents for use in the invention are those whichinteract with ionized or ionizable groups in a sample to improveresolution including both cationic and anionic ion-pairing reagents.Cationic ion-pairing agents for use in the invention include amines suchas lower alkyl primary, secondary, and tertiary amines (e.g.,triethylamine (TEA)), ammonium salts such as lower trialkylammoniumsalts of organic or inorganic acids (e.g., triethylammonium acetate) andlower quaternary ammonium salts such as tetrabutylammonium phosphate.Anionic ion-pairing agents include perfluorinated carboxylic acids.Herein, “lower alkyl” refers to an alkyl group of one to six carbonatoms, as exemplified by methyl, ethyl, n-butyl, i-butyl, t-butyl,isoamyl, n-pentyl, and isopentyl.

[0069] The hydrophobicity of the ion-pairing agent will vary dependingupon the nature of the desired separation. For example,tetrabutylammonium phosphate is considered a strongly hydrophobic cationwhile triethylamine is a weak hydrophobic cationic ion-pairing reagent.Generally, preferred ion-pairing agents are cationic in nature for acidsand anionic for bases. One such preferred ion-pairing agent for use inthe invention is triethylammonium acetate (TEAA).

[0070] In accordance with the present invention, preferred solvents foruse in the mobile phase are organic solvents. The organic solvent,occasionally referred to as an organic modifier, is any organic (e.g.,non-aqueous) liquid suitable for use in the chromatographic separationmethods of the present invention. Generally, the organic solvent is apolar solvent (e.g., more polar than the stationary support) such asacetonitrile, methanol, ethanol, ethyl acetate, and 2-propanol. Apreferred solvent is acetonitrile.

[0071] The pH of the mobile phase will vary depending upon theconcentrations of various components. For separation of nucleic acidsamples such as RNA or DNA molecules, using temperature to effectpartial denaturation of the nucleic acid, the pH of the mobile phase istypically maintained within a range of about 7 to about 9. Preferably,the mobile phase is maintained at a pH of about 7.5.

[0072] In an alternate embodiment, the pH of the mobile phase isadjusted to effect partial denaturation of the heteroduplex molecules ina mixture of homoduplexes and heteroduplexes to allow separation anddetection of the heteroduplex molecules. In using chemical means toeffect heteroduplex denaturation, the pH may be adjusted by addition ofeither base (e.g., sodium hydroxide or urea to a pH of around about 8)or acid (e.g., triethylamine and acetic acid at a pH of about 8) underconditions effective to partially denature the heteroduplex moleculesand which do not degrade the nucleic acids present in the sample noradversely affect the integrity of the stationary phase. In such cases,sample elution may be carried out at temperatures less than about 50° C.

[0073] The concentration of the mobile phase components will varydepending upon the nature of the separation to be carried out. Themobile phase composition may vary from sample to sample and during thecourse of the sample elution. The concentration of the ion-pairing agentin the mobile phase is preferably less than about 1.0 molar, morepreferably within a range of about 50 mM to about 200 mM, and mostpreferably at a concentration of about 100 millimolar. The mobile phasepreferably includes less than about 40% by volume of an organic solvent.

[0074] Samples are typically eluted by starting with an aqueous ormostly aqueous mobile phase containing an ion-pairing agent andprogressing to a mobile phase containing increasing amounts of anorganic solvent. Any of a number of gradient profiles and systemcomponents may be used to achieve the denaturing conditions of thepresent invention. One such exemplary gradient system in accordance withthe invention is a linear binary gradient system composed of 0.1 molartriethylammonium acetate, 0.1 millimolar ethylenediaminetetraacetic acid(EDTA), and 25% acetonitrile in a solution of 0.1 molar triethylammoniumacetate and 0.1 millimolar EDTA. The EDTA is typically used when thereversed phase support is a silica-based material to prevent DNAadsorbing to the silica and/or metal chelation.

[0075] One way to achieve the denaturing conditions of the presentinvention (e.g., effective to partially denature heteroduplexes) is tomodulate column temperature. A column temperature typically of about 50°C. to about 65° C. is preferred for resolving heteroduplex moleculesfrom their corresponding homoduplex molecules. The optimal columntemperature will depend upon the putative sequence (base composition) ofthe nucleic acid samples to be separated and the particular detectabletag. Thus, the choice of stationary phase, the choice of mobile phase,pH, flow rate, and the like, and in many cases, will be determinedempirically. Ideally, in cases with known sequence, a suitable columntemperature may be calculated.

[0076] Detection

[0077] By incorporating a detectable tag, a mixture containing aplurality of labeled nucleic acid samples can be applied to a first endof a chromatograpy column containing a stationary phase, preferably inthe presence of a mobile phase. These samples are run substantiallysimultaneously through the column under conditions to partially denaturethe nucleic acid molecules and separate the samples. Upon elution, thenucleic acid species of each of the separately labeled nucleic acidsample pass through the detection zone substantially simultaneously,each sample generating a specific signal which is spectrally resolvablefrom the specific signals of the other nucleic acid samples.Consequently, each nucleic acid sample generates a chromatogram that canbe reconstructed to represent the elution pattern of the individualnucleic acid species in this sample. This chromatogram is distinct andindependent of the similarly obtained chromatograms for the othernucleic acid samples that were injected into the system.

[0078] A multiwavelength spectral (preferably, fluorescence) detector ispreferably operably attached to a second end of the chromatographycolumn. The detector can be used to excite the detectable tags at onewavelength and detect emissions as multiple wavelengths, or excite atmultiple wavelengths and detect at one emission wavelength.Alternatively, the sample can be excited using “zero-order” excitationin which the full spectrum of light (e.g., from xenon lamp) illuminatesthe flow cell. Each compound can absorb at its characteristic wavelengthof light and then emit maximum fluorescence. The multiple emissionsignals can be monitored independently. Preferably, a suitable detectorcan be programmed to detect more than one excitation emission wavelengthsubstantially simultaneously, such as that commercially available underthe trade designation HP1100 (G1321A), from Hewlett Packard, Wilmington,DE. Thus, the labeled nucleic acid samples eluted from the stationaryphase can be detected at programmed emission wavelengths at variousintervals during elution.

[0079] A preferred detector can be programmed to detect at least two,and preferably at least four different excitation or emissionwavelengths simultaneously. Thus, PCR products from at least twoindividuals, each labeled with a different fluorescent tag, can beco-injected, increasing throughput by a factor of at least two. Sincethe detector has time resolution capabilities, the initial portion ofthe chromatogram can be scanned for at least two emission wavelengthsand the later portion of the chromatogram can be scanned for a secondset of at least two emission wavelengths. By combining size of thenucleic acid molecules and fluorescence multiplexing, the throughput ofthe system can be increased at least 8-fold, e.g., four dyes at two sizeranges.

[0080] In carrying out the separation method of the present invention, avariety of factors may influence detection resolution. While it is notpossible to determine an ideal set of conditions suitable for analyzingall possible nucleic acid fragments by the present method, conditionscan be determined empirically to affect detection resolution.

[0081] In carrying out the method of the invention, a mixture containingnucleic acid samples (e.g., PCR amplification products) to be analyzedis typically injected and pre-mixed with the mobile phase prior toelution on the solid support. The sample is then contacted directly withthe stationary phase, or alternatively, is passed through a“pre-conditioning” tubing or pre-column to allow the sample and mobilephase to equilibrate before contact with the solid support.

[0082] For example, the mobile phase components are introduced into amixer inside the column oven and mixed prior to contact with the sample.Alternatively, the mobile phase components may be mixed at ambienttemperature and contacted with the sample injector, also maintained atambient temperature outside of the column oven. Additionally, themixture can be injected into the mobile phase, pre-equilibrated to thetemperature of the column. In this manner, a near-direct connectionbetween the column and the injector is provided to minimize diffusionand enhance sample resolution.

[0083] Alternately, when utilizing a low-pressure system, sample mixingtypically occurs at ambient temperature. In instances in which theautosampler does not provide for heating the injection port to columntemperature, standard HPLC tubing (e.g., 0.005-0.01 inch in diameter)may be positioned between the injector and the column, to pre-heat themobile phase and induce partial denaturation of the DNA sample.

[0084] Another factor which affects the parameters to be selected forcarrying out the separation method of the invention is the compositionof the putative sequence of the PCR amplification products to beanalyzed. In this respect, for mixtures containing a polymorphic siteflanked by a GC-rich region, higher temperatures may be required todetect the polymorphism.

[0085] Genotyping

[0086] In genotyping two alleles of a polymorphic site of interest, anexemplary method includes generating two labeled nucleic acid samples(e.g., PCR products) where each of the samples includes one of theputative alleles and a detectable tag, thereby forming referencegenotypes. A labeled mixture is formed by mixing the two labeled samplesin a ratio of about 1:1. An unlabeled nucleic acid sample (e.g., PCRproduct) is generated from a sample of interest (e.g., the analyte orunknown entity being evaluated), which contains allele 1 and/or allele2. A hybrid mixture containing labeled and unlabeled nucleic acid isformed by mixing the labeled mixture with the unlabeled nucleic acidsample. Preferably, the unlabeled sample is present in excess relativeto the labeled mixture, preferably, in an excess of at least about 5:1,and more preferably, at least about 10:1.

[0087] The hybrid mixture is then denatured and reannealed attemperatures depending on the particular nucleic acids under study(e.g., by heating at a temperature up to about 99° C. for 5 minutesfollowed by cooling to 45° C. over a period of about 30 minutes). Theunlabeled nucleic acid sample is used in excess of the labeled mixtureso that one labeled nucleic acid strand preferentially hybridizes to anunlabeled nucleic acid strand rather than to another labeled nucleicacid strand. For the purposes of illustration, the labeled nucleic acidsample (e.g., PCR product) of allele 1 is referred to as “Label I” andthat of allele 2 is referred to as “Label 2.” The following tableillustrates the combinations that are possible as a result of denaturingand reannealing. TABLE 1 Putative Alleles Present in Result AfterReannealing With Sample Unlabeled PCR Product Two copies of allele 1Label 1 forms only homoduplex Label 2 forms only heteroduplex One copyof allele 1 Label 1 forms both a homoduplex and One copy of allele 2heteroduplex Label 2 forms both a homoduplex and heteroduplex Two copiesof allele 2 Label 1 forms only heteroduplex Label 2 forms onlyhomoduplex

[0088] By generating a denaturing liquid chromatogram using the abovemixture as a sample and utilizing a fluorescence detector duringelution, the genotype can be determined by comparing the results of theelution profiles of each label to the above table.

[0089] Haplotyping

[0090] The method for genotyping, illustrated above, can be expanded todetermine haplotype. For the purposes of illustration, alleles 1 and 2exist at one polymorphic site and alleles 3 and 4 exist at a secondpolymorphic site linked to the first. Labeled samples of each haplotypeare preferably generated by incorporating a detectable tag such thateach of the 4 haplotypes contain a tag having a different detectablesignal. As described above, once the 4 labeled samples are obtained, amixture of the labeled samples is formed. Preferably, they are mixed ina ratio such that the signal (e.g., fluorescent signal) per product isapproximately equal. This can be done by adjusting the relativeconcentration of each sample in the mixture. The sample of interest(analyte) is generated in an unlabeled form. A hybrid mixture includingthe mixture of labeled samples and the unlabeled sample is formed bymixing the unlabeled sample in a significant excess relative to thelabeled samples. Preferably, the ratio of unlabeled to labeled samplesis at least about 5:1, and more preferably, at least about 10:1.

[0091] The hybrid mixture is then denatured and reannealed attemperatures depending on the particular nucleic acids under study. Theunlabeled sample is used in excess of the labeled samples so that eachlabeled strand preferentially hybridizes to an unlabeled strand ratherthan to another labeled strand. For the purposes of illustration, assumethat the labeled strands contain the haplotypes and the labels listed inthe table below. TABLE 2 Putative Haplotype Haplotype Designation Labelin PCR Product Allele 1 and Allele 3 Hap 1 Label 1 Allele 1 and Allele 4Hap 2 Label 2 Allele 2 and Allele 3 Hap 3 Label 3 Allele 2 and Allele 4Hap 4 Label 4

[0092] Next, using the four labeled reference haplotype samples, thefollowing duplex formation of each haplotype in a sample containing oneof the four reference haplotypes can be deduced. In each of the caseswhere a heterozygote is deduced, a heteroduplex is formed, either withone or two mismatches. TABLE 3 Duplex Duplex Duplex Duplex Sample formedwith formed with formed with formed with Haplotype Hap 1 Hap 2 Hap 3 Hap4 Hap 1 Homoduplex Heteroduplex Heteroduplex Heteroduplex (1 mismatch)(1 mismatch) (2 mismatches) Hap 2 Heteroduplex Homoduplex HeteroduplexHeteroduplex (1 mismatch) (2 mismatches) (1 mismatch) Hap 3 HeteroduplexHeteroduplex Homoduplex Heteroduplex (1 mismatch) (2 mismatches) (1mismatch) Hap 4 Heteroduplex Heteroduplex Heteroduplex Homoduplex (2mismatches) (1 mismatch) (1 mismatch)

[0093] The possible combinations that can result from a sample witheither one or two of the four unique haplotypes are summarized in thefollowing table. TABLE 4 Duplex Duplex Duplex Duplex Sample formed withformed with formed with formed with Haplotype Hap 1 Hap 2 Hap 3 Hap 4Hap 1/Hap 1 Homoduplex Heteroduplex Heteroduplex heteroduplex (1mismatch) (1 mismatch) (2 mismatches) Hap 1/Hap 2 Homoduplex andHomoduplex and Heteroduplex Heteroduplex Heteroduplex Heteroduplex (1mismatch) and (1 mismatch) and (1 mismatch) (1 mismatch) HeteroduplexHeteroduplex (2 mismatches) (2 mismatches) Hap 1/Hap 3 Homoduplex andHeteroduplex Homoduplex and Heteroduplex Heteroduplex (1 mismatch) andHeteroduplex (1 mismatch) and (1 mismatch) Heteroduplex (1 mismatch)Heteroduplex (2 mismatches) (2 mismatches) Hap 1/Hap 4 Homoduplex andHeteroduplex Heteroduplex Homoduplex and Heteroduplex (1 mismatch) (1mismatch) Heteroduplex (2 mismatches) (2 mismatches) Hap 2/Hap 2Heteroduplex Homoduplex Heteroduplex Heteroduplex (1 mismatch) (2mismatches) (1 mismatch) Hap 2/Hap 3 Heteroduplex Homoduplex andHomoduplex and Heteroduplex (1 mismatch) Heteroduplex Heteroduplex (1mismatch) (1 mismatch) (2 mismatches) Hap 2/Hap 4 HeteroduplexHomoduplex and Heteroduplex Homoduplex and (1 mismatch) and Heteroduplex(1 mismatch) and Heteroduplex Heteroduplex (1 mismatch) Heteroduplex (1mismatch) (2 mismatches) (2 mismatches) Hap 3/Hap 3 HeteroduplexHeteroduplex Homoduplex Heteroduplex (1 mismatch) and (2 mismatches) (1mismatch) Heteroduplex (2 mismatches) Hap 3/Hap 4 HeteroduplexHeteroduplex Homoduplex and Homoduplex and (2 mismatches) (1 mismatch)and Heteroduplex Heteroduplex Heteroduplex (1 mismatch) (1 mismatch) (2mismatches) Hap 4/Hap 4 Heteroduplex Heteroduplex HeteroduplexHomoduplex (2 mismatches) (1 mismatch) (1 mismatch)

[0094] Because each of the four labeled nucleic acid samples (e.g., PCRproducts) corresponding to the four reference haplotypes contains adifferent label, a unique elution chromatagraphic pattern in each labelwill be associated 5 with any one of the ten combination haplotypeslisted above. Because the unlabeled nucleic acid sample is included inthe hybrid mixture in significant excess of the labeled samples, a verysmall amount of labeled/labeled duplex would be expected to anneal andthus, a very small amount of labeled/labeled duplex is likely to bedetected. Additionally, if only one strand of the labeled nucleic acidsample actually includes the label, then only one heteroduplex would bedetected rather than two if both strands were labeled. However, it maybe advantageous to label both strands of a labeled nucleic acid samplebecause two different heteroduplexes may be detected, which may provideconfirmatory information in the measurement.

EXAMPLES

[0095] The objects, features and advantages of the present inventionillustrated in the following examples, which incorporate particularmaterials and amounts, should not be construed to unduly limit thisinvention. All materials are commercially available unless otherwisestated or apparent. All parts, percentages, ratios, etc., in theexamples are by weight unless otherwise indicated.

[0096] In the spectrum shown in FIG. 1, two samples, each consisting ofequal mixtures of a “wild-type” sequence and an A to G variant atsequence position Y were labeled with different fluorescent dyes andanalyzed simultaneously by fluorescence multiplexed DHPLC. Theindividual signals were separated and resolved spectrally. In FIG. 1A,the control homoduplex sample labeled with Cy3 and Cy5 cyan dyes(Amersham-Pharmacia Biotech.) was predominantly two well resolved peaks.In the lower panel (FIG. 1B), test sample consisting of both homo- andheteroduplex samples labeled both Cy3 and CyS shows a distinctlydifferent pattern indicating the presence of a polymorphism.

[0097] The individual samples were prepared by PCR amplifying the twoindividual templates (termed 209A and 209G) with one of the PCR primersfluorescently labeled. In one case the primer was labeled with a Cy3 dye(Excitation =550 nm; Emission=570 nm), in the other case the primer waslabeled with a Cy5 dye (Excitation=649 nm; Emission=670 nm). Followingamplification, the Cy3 labeled 209A and 209G amplicons were mixed inequal proportions and placed in a separate PCR tube. The Cy5 labeled209A and 209G samples were mixed in equal proportions and placed in aseparate tube. These two mixtures were then heat treated to producemixtures of homo- and heteroduplexes by heating to 99° C. for 5 minutesand gradually cooling to 45° C. over 60 minutes and then held at 4° C.for 90 minutes. The resulting Cy3 and Cy5 labeled homo- and heteroduplexmixtures were then pooled and used for a single simultaneous analysis byfluorescence multiplexed DHPLC.

[0098] It should be noted that adding a tag may change the optimaltemperature for partial denaturation, so that the operating temperaturemay need to be adjusted from the predicted value. Also, adding a tag maychange the optimal HPLC gradient for resolution, so the separationconditions may need to be adjusted from the predicted value.

[0099] All DHPLC analytical analyses were performed using aHewlett-Packard HP 1100 HPLC with Binary Pump, thermostatted autosamper,thermostatted column oven, Diode Array UV/VIS detector and FluorescenceDetector. This system is unmodified and commercially available exceptfor the addition of an aluminum block around the HPLC column to helpmaintain constant temperature. The HPLC column consisted of a 75 mm×2.1mm I.D., 3.5 μm Eclipse dsDNA column with a guard column. For thisparticular example, the column temperature was held at 54° C. Mobilephase A consisted of 100 mM triethylammonium acetate, 0.1 mM EDTA.Mobile phase B consisted of 25% acetonitrile, 100 mM triethylammoniumacetate, and 0.1 mM EDTA. The initial mobile phase was 45% B at 0.4ml/minute. Following injection of 2 μL of sample, the mobile phasegradient ramped mobile phase to 60% B in 0.1 minute followed by a rampto 80% B in 10 minutes. By this time the samples eluted from the columnand the mobile phase was changed to 100% B and held for 1 minute toclean up the column and then reequilibrated at 45% B for 5 minutes. Thesamples eluting from the column entered the fluorescence detector with azero-order broad band excitation and emission monitored at 570 nm forCy3 and 670 nm for Cy5.

[0100] Patents, patent applications, and publications disclosed hereinare hereby incorporated by reference as if individually incorporated. Itis to be understood that the above description is intended to beillustrative, and not restrictive. Various modifications and alterationsof this invention will become apparent to those skilled in the art fromthe foregoing description without departing from the scope and thespirit of this invention, and it should be understood that thisinvention is not to be unduly limited to the illustrative embodimentsset forth herein.

What is claimed is:
 1. A method for separating nucleic acid samples in atest mixture, the method comprising: providing a test mixture comprisinga plurality of nucleic acid samples, wherein each sample is labeled witha spectrally detectable tag; applying the test mixture to a reversedphase support; eluting the test mixture under partially denaturingconditions to separate at least one of the nucleic acid samples from thetest mixture; and detecting spectrally resolved signals produced by thenucleic acid samples labeled with the detectable tags.
 2. The method ofclaim 1 wherein the nucleic acid samples comprise PCR heteroduplex andhomoduplex products.
 3. The method of claim 1 wherein applying a mixtureto a reversed phase support comprises applying the mixture to a highperformance liquid chromatography column comprising a reversed phasesupport.
 4. The method of claim 1 wherein each detectable tag comprisesa fluorescent dye.
 5. The method of claim 4 wherein providing a testmixture comprises: combining a different fluorescent dye with eachnucleic acid sample to form a labeled nucleic acid sample; and combiningthe labeled nucleic acid samples to form a test mixture.
 6. The methodof claim 5 wherein combining a different fluorescent dye with eachnucleic acid sample comprises combining a different fluorescent dye witheach nucleic acid sample during polymerase chain reaction amplificationof the nucleic acid sample.
 7. The method of claim 1 wherein the nucleicacid sample comprises DNA or RNA.
 8. The method of claim 1 whereineluting the test mixture under partially denaturing conditions compriseseluting with a mobile phase comprising an ion-pairing agent and anorganic solvent.
 9. The method of claim 1 wherein the partiallydenaturing conditions comprise a temperature of about 50° C. to about80° C.
 10. The method of claim 1 wherein detecting spectrally resolvedsignals comprises use of a spectrophotofluorometric HPLC detector. 11.The method of claim 1 wherein the tag is a spectrally detectable tagselected from the group of an electromagnetic tag and an electrochemicaltag.
 12. The method of claim 11 wherein the tag is a spectrallydetectable tag selected from the group of a spectrophotometric tag and aspectrofluorometric tag.
 13. The method of claim 1 wherein detectingspectrally resolved signals produced by the nucleic acid samples labeledwith the detectable tags comprises passing the separately labelednucleic acid samples through a detection zone of a detectorsubstantially simultaneously, each sample generating a specific signalwhich is spectrally resolved from the specific signals of the othernucleic acid samples.
 14. The method of claim 13 wherein the detectorexcites the detectable tags at one wavelength and detects emissions atmultiple wavelengths.
 15. The method of claim 13 wherein the detectorexcites the detectable tags using zero-order excitation.
 16. A methodfor detecting genotypic variations, the method comprising: providing apre-mixture comprising one unlabeled nucleic acid sample and two or morereference genotypes comprising labeled nucleic acid, wherein eachlabeled nucleic acid is labeled with a different detectable tag and theunlabeled nucleic acid is present in an excess amount relative to thetotal amount of labeled nucleic acid; denaturing and reannealing thepre-mixture to form a test mixture comprising labeled/unlabeled nucleicacid duplexes; applying the test mixture to a reversed phase support;eluting the test mixture under partially denaturing conditions toseparate at least one of the labeled/unlabeled nucleic acid duplexesfrom the test mixture; detecting spectrally resolved signals produced bythe labeled nucleic acid to define an elution profile for each tag; anddeducing from the elution profiles the composition of the unlabelednucleic acid sample.
 17. The method of claim 16 wherein the tag is aspectrally detectable tag selected from the group of an electromagnetictag and an electrochemical tag.
 18. The method of claim 17 wherein thetag is a spectrally detectable tag selected from the group of aspectrophotometric tag and a spectrofluorometric tag.
 19. The method ofclaim 16 wherein the labeled and unlabeled nucleic acid comprise PCRproducts.
 20. The method of claim 16 wherein applying the test mixtureto a reversed phase support comprises applying the mixture to a highperformance liquid chromatography column comprising a reversed phasesupport.
 21. The method of claim 16 wherein each spectrally detectabletag comprises a fluorescent dye.
 22. The method of claim 16 whereindetecting spectrally resolved signals produced by the labeled nucleicacid comprises passing the separately labeled nucleic acid samplesthrough a detection zone of a detector substantially simultaneously,each sample generating a specific signal which is spectrally resolvedfrom the specific signals of the other nucleic acid samples.
 23. Themethod of claim 22 wherein the detector excites the detectable tags atone wavelength and detects emissions at multiple wavelengths.
 24. Themethod of claim 22 wherein the detector excites the detectable tagsusing zero-order excitation.
 25. A method for determining haplotypes,the method comprising: providing a pre-mixture comprising one unlabelednucleic acid sample and four or more reference haplotypes comprisinglabeled nucleic acid, wherein each labeled nucleic acid sample includestwo or more alleles, each reference haplotype is labeled with adifferent detectable tag, and the unlabeled nucleic acid is present inan excess amount relative to the total amount of labeled nucleic acid;denaturing and reannealing the pre-mixture to form a test mixturecomprising labeled/unlabeled nucleic acid duplexes; applying the testmixture to a reversed phase support; eluting the test mixture underpartially denaturing conditions to separate at least one of thelabeled/unlabeled nucleic acid duplexes from the test mixture; detectingspectrally resolved signals produced by the labeled nucleic acid todefine an elution profile for each tag; and deducing from the elutionprofiles the composition of the unlabeled nucleic acid sample.
 26. Themethod of claim 25 wherein the tag is a spectrally detectable tagselected from the group of an electromagnetic tag and an electrochemicaltag.
 27. The method of claim 26 wherein the tag is a spectrallydetectable tag selected from the group of a spectrophotometric tag and aspectrofluorometric tag.
 28. The method of claim 27 wherein eachspectrally detectable tag comprises a fluorescent dye.
 29. The method ofclaim 25 wherein detecting spectrally resolved signals produced by thelabeled nucleic acid comprises passing the separately labeled nucleicacid samples through a detection zone of a detector substantiallysimultaneously, each sample generating a specific signal which isspectrally resolved from the specific signals of the other nucleic acidsamples.
 30. The method of claim 29 wherein the detector excites thedetectable tags using zero-order excitation.